In the field of optical imaging for biological diagnostics, if specific detection other than contact imaging is focused on, two methods are commonly used. These methods are flow cytometry and fluorescence molecular imaging.
Flow cytometry is a powerful technique that counts, characterizes and sorts the various cells that cut the light beam of a laser. By analyzing the diffraction patterns created it is possible to determine the dimensions of the cells. Fluorescence measurements furthermore allow the various families of bacteria to be distinguished. The drawback of this technique is that it requires expensive and complicated equipment. Another drawback is that the solid angle scanned is relatively small, limiting the field that can be investigated.
Fluorescence molecular imaging is a method widely used in biological diagnostics because it is extremely effective. Fluorescence measurements are sensitive to single events: using fluorescent labels it is possible to detect individual molecules using a microscope. To obtain good results it is necessary to completely separate the energy for exciting the fluorescent molecules—called the “excitation” energy—from the energy emitted by the fluorescent molecules—called the “emission” energy. Although excellent filters exist at the present time, they constrain the light beams, in particular requiring them to have small beam-apertures. Consequently, the optical systems that this method uses are complicated and bulky. Fluorescence imaging also requires prior addition of a fluorophore to the medium to be analyzed, making the process invasive.
Thus, it is desired to replace these complicated and costly techniques with noninvasive contact imaging devices having extended fields of observation.
These techniques are being developed further and further because they allow cells, bacteria or more generally micron-sized particles to be detected without requiring the aforementioned advanced optical systems. A schematic of a contact imaging device is shown in FIG. 1. This device comprises a light source 1, possibly a small source, for example a light-emitting diode, a diaphragm 2 limiting the aperture of the source and an imager or sensor 3, which may be a matrix of CCD (charge-coupled device) or CMOS (complementary metal oxide semiconductor) photosites. Such imagers generally comprise microlenses associated with each photosite. The diaphragm 2 is not essential, but its presence is advantageous. Inserted between this matrix 3 and the light source 1 is a transparent microscope slide 4 that carries the object 5 to be studied.
This object is a solution containing micron-sized particles—these particles may be biological particles such as cells or bacteria or other particles such as microspheres. The droplet analyzed rests on the transparent slide 4 and its meniscus is in contact with the ambient gas, which gas may be air. The matrix 3 is connected to an image display and/or processing system, not shown in FIG. 1. The distance separating the diode 1 from the object-carrying slide 4 is preferably greater than 1 cm and may be, for example, a few cm, typically between 2 cm and 5 cm. The distance separating the object from the surface of the sensor is between 0.1 mm and 2 mm. Although this is referred to as contact imaging, the object to be studied is not placed in direct contact with the sensor but at the distance indicated above. The slide is made of a transparent material such as silica or quartz and its thickness varies between a few tens of microns and 1 mm. This very simple device, without magnifying optics, may, in certain cases, be an alternative to the conventional optical counting methods such as flow cytometry, high-resolution optical microscopy or fluorescence molecular imaging.
Over the last few years, several teams have obtained impressive results using contact imaging. Thus, a team based at the American university UCLA (University of California, Los Angeles) used contact imaging to detect and identify bacteria. This method is described in the following publication: “Lensfree holographic imaging for on-chip cytometry and diagnostics” by Sungkyu Seo et al., The Royal Society of Chemistry, Dec. 5, 2008-2009, 9, 777-787. In the devices described in that publication, the bacteria are placed in a liquid between two plates, the assembly being put on a matrix of photosites. A monochromatic illumination source is filtered by a 100-μm diameter diaphragm so as to obtain good spatial coherence. Thus, at the matrix of photosites, a diffraction pattern is obtained for each immersed cell. According to the authors, the diffraction patterns obtained are sufficiently well-resolved and distinct from one species to another that specific counting of the various bacteria is possible.
The method proposed by the UCLA team is elegant. However, it has a drawback—it requires the use of high-sensitivity CCD sensors that are necessarily costly. Thus, the sensors used were Kodak Kai-10002 high-sensitivity CCD sensors.
If standard sensors, such as low-cost CMOS or CCD sensors, are used, it is still possible to observe the diffraction patterns of the various 1 μm diameter micron-sized particles such as silanol microspheres, latex microspheres or E. Coli bacteria. However, the detection efficiency is very low, at best about 1%. Thus, FIG. 2 shows the signal S obtained along an axis x of the sensor that passes through the center of a particle P. The signal S shows here the gray level along the ordinate axis measured by various pixels, the pixels forming the abscissa axis, i.e. FIG. 2 shows a profile. It may be seen that the signal-to-noise ratio is very low, barely sufficient to allow the particle to be detected.